SOP for Calibration of UV-Vis Spectrophotometer

Learn how to write a standard procedure SOP for calibration of UV-Vis Spectrophotometer in quality control laboratory.

1. PURPOSE

To outlines the steps for the calibration of the UV-Vis Spectrophotometer to ensure accurate and precise measurement of absorbance in pharmaceutical applications. Proper calibration is critical to obtain reliable analytical results that meet regulatory and quality control standards.

2. SCOPE

This SOP is applicable to the UV-Vis Spectrophotometer used in the Quality Control (QC) Laboratory for the measurement of absorbance, transmittance, and related analytical parameters for pharmaceutical products.

3. RESPONSIBILITIES

3.1 Executive / Officer / Analyst, Quality control: Perform the calibration procedure according to the steps outlined in this SOP.

3.2 Quality Control (QC) Manager: Supervise the calibration process and ensure that the procedure is followed.

3.3 Maintenance Personnel: Conduct routine maintenance and repairs of the spectrophotometer as needed.

4. ACCOUNTABILITY

Head of the Quality Assurance Department

5. ASSOCIATED DOCUMENTS / REFERENCES

5.1 UV-Visible Spectrophotometer User Manual (Manufacturer)

5.2 USP <857> UV-Visible Spectroscopy

5.3 ICH Q2 (R1) Validation of Analytical Procedures: Text and Methodology

6. PRECAUTIONS

6.1 Wear appropriate personal protective equipment (PPE), such as gloves and safety goggles, when handling reagents.

6.2 Follow the manufacturer’s safety guidelines when working with reagents and the apparatus.

6.3 Temperature-controlled environment (20°C to 25°C)

7. PROCEDURE

7.1 Equipment and Materials

7.1.1 UV-vis Spectrophotometer

7.1.2 Calibration Standards: Potassium Dichromate (K₂Cr₂O₇) Solution (1.0 mg/mL), Holmium Oxide (Ho₂O₃) Solution (1.0 mg/mL)

7.1.3 Quartz Cuvettes: Clean, dry, and free of scratches.

7.1.4 Deionized (DI) Water

7.1.5 Lint-free wipes or soft cloths for cleaning the cuvettes

7.2 Pre-Calibration Preparation

7.2.1 Ensure the UV-Vis spectrophotometer is in proper working condition.

7.2.2 Perform a visual inspection of the instrument, checking for any visible signs of damage or wear.

7.2.3 Warm-Up the Spectrophotometer:

7.2.4 Switch on the UV-vis spectrophotometer and allow it to warm up for at least 30 minutes to stabilize the lamp and ensure accurate readings.

7.2.5 Clean the quartz cuvettes with deionized water and dry them with a lint-free cloth or air dry.

7.2.6 Ensure there are no fingerprints, dust, or residues on the cuvettes.

7.3 Blank Calibration

7.3.1 Prepare the Blank solution

7.3.2 Use deionized water as the blank solution for calibration.

7.3.3 Fill a clean quartz cuvette with deionized water.

7.3.4 Insert the cuvette with deionized water into the spectrophotometer’s sample compartment.

7.3.5 Close the lid of the sample compartment to avoid interference from ambient light.

7.3.6 Set the spectrophotometer to the desired wavelength (usually 254 nm for UV calibration).

7.3.7 Press the “Zero” or “Blank” button on the spectrophotometer to set the baseline absorbance to zero.

7.4 Absorbance Calibration Using Potassium Dichromate

7.4.1 Prepare or procure a potassium dichromate solution of 1.0 mg/mL concentration in deionized water.

7.4.2 Fill a clean quartz cuvette with the potassium dichromate solution.

7.4.3 Insert the cuvette into the sample compartment and record the absorbance at wavelengths 235 nm, 257 nm, and 313 nm.

7.4.4 The absorbance values at the specified wavelengths should match the reference values provided in the instrument’s manual. If there is a discrepancy, perform the necessary adjustments as per the manufacturer’s guidelines.

7.5 Wavelength Calibration Using Holmium Oxide Solution

7.5.1 Prepare or procure a holmium oxide solution of 1.0 mg/mL concentration in deionized water.

7.5.2 Fill a clean quartz cuvette with the holmium oxide solution.

7.5.3 Insert the cuvette into the sample compartment.

7.5.4 Record the absorbance at specific wavelengths (e.g., 241 nm, 287 nm, 333 nm, 361 nm, 420 nm).

7.5.5 Compare the recorded absorbance values against the standard reference data provided for the holmium oxide solution. Any deviations must be addressed by adjusting the instrument’s wavelength calibration settings.

7.6 Linearity Test

7.6.1 Measure the absorbance of standard solutions with known concentrations (e.g., 0.1 mg/mL, 0.5 mg/mL, 1.0 mg/mL) of a reference compound.

7.6.2 Plot absorbance against concentration and verify the linearity of the response.

7.6.3 The absorbance should show a linear relationship with the concentration. If the result is nonlinear, perform a re-calibration or check the instrument for faults.

7.7 Post-Calibration Cleaning

7.7.1 After calibration, clean the cuvettes using deionized water and dry them properly.

7.7.2 Switch off the UV-vis spectrophotometer and store it in a clean, dry place when not in use.

7.8 Documentation

7.8.1 Record all calibration results in the Calibration Logbook.

7.8.2 Ensure the following details are documented:

7.8.3 Date and time of calibration

7.8.4 Name of the person performing the calibration

7.8.5 Calibration results and any deviations

7.8.6 Actions taken to resolve discrepancies (if any)

7.8.7 Signature of the QC Manager and the technician

7.9 Frequency of Calibration

7.9.1 Routine Calibration: The UV-vis spectrophotometer must be calibrated at least once every 6 months.

7.9.2 Verification Calibration: After any maintenance, lamp change, or significant changes in environmental conditions, the spectrophotometer should be recalibrated.

8. ABBREVIATIONS / DEFINITIONS

8.1 UV-visible Spectrophotometer: An ultraviolet-visible (UV-Vis spectrophotometer) used to measure absorbance and transmittance of samples in the ultraviolet and visible light regions.

8.2 Calibration: The process of adjusting and verifying the instrument’s accuracy and performance to ensure it provides correct and consistent results.

8.3 SOP: Standard Operating Procedure

8.4 ICH: International Council for Harmonization

8.5 USP: United States Pharmacopoeia

8.6 UV: Ultraviolet

8.7 QC: Quality Control

8.7 PPE: Personal Protective Equipment

9. ANNEXES

9.1 Annex-1: Log Book for Calibration of UV-Vis Spectrophotometer

10. Revision History

Version	Date	Description of Change	Reviewed By	Approved By
1.0